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HLA-G is a HLA class Ib antigen that possesses immunomodulatory properties. HLA-G-expressing CD4+ and CD8+ T lymphocytes, NK cells, monocytes, and dendritic cells with immunoregulatory functions are present in small percentages of patients with physiologic conditions. Quantitative and qualitative derangements of HLA-G+ immune cells have been detected in several conditions in which the immune system plays an important role, such as infectious, neoplastic, and autoimmune diseases as well as in complications from transplants and pregnancy. These observations strongly support the hypothesis that HLA-G+ immune cells may be implicated in the complex mechanisms underlying the pathogenesis of these disorders.

BackgroundSystemic sclerosis (SSc) is a complex disease characterised by immune abnormalities, vascular damage and fibrosis. Human leukocyte antigen-G (HLA-G) is a non-classic class I major histocompatibility complex (MHC) molecule expressed on different cell lineages in both physiological and pathological conditions and detectable in soluble forms (sHLA-G1 and HLA-G5 shed and secreted isoform, respectively). Several immunomodulatory functions have been attributed to both membrane-bound and soluble HLA-G molecules. HLA-G is expressed on extravillous cytotrofoblast, in placenta but also in a few normal tissues, solid tumours, transplanted organs and virally infected cells. Soluble form (sHLA-G) derives from shedding of cleaved surface isoforms (sHLA-G1) or secretion of soluble isoforms (HLA-G5). Immunosuppressive functions have been attributed to both membrane HLA-G (mHLA-G) and sHLA-G.ObjectivesThe aims of the present study were: 1) to determine the serum levels of sHLA-G molecules in a cohort of SSc patients with the limited or diffuse form of the disease; 2) to correlate sHLA-G levels with TGF-β; 3) to evaluate the expression of HLA-G in peripheral blood mononuclear cells (PBMC).MethodsThirtyfive patients (28 females/7 males, age 40–89 years) with diffuse SSc (dSSc, n. 12) or limited SSc (lSSc, n. 23) and 40 healthy sex and age matched controls were enrolled. Plasma sHLA-G, sHLA-G1 and HLA-G5 levels were determined by immunoenzymatic assays. mHLA-G expression in peripheral blood mononuclear cells (PBMC) was evaluated by flow cytometry.ResultsThe plasma levels of sHLA-G were higher in SSc patients (444.27±304.84 U/ml) compared to controls (16.74±20.58 U/ml) (p<0.0001). The plasma levels of TGF-β were higher in SSc patients (18937±15217 pg/ml) compared to controls (11099±6081 pg/ml; p=0.003) and a significant correlation was found between TGFβ and the plasma levels of total sHLA-G (r: 0.65; p<0.01), sHLA-G1 (r: 0.60; p=0.003) and HLA-G5 (r: 0.47; p=0.02). The percentage of HLA-G-positive monocytes (0.98±1.72), CD4+ (0.37±0.68), CD8+ (2.05±3.74) and CD4 +CD8+double positive cells (14.53±16.88) was higher in SSc patients than in controls (0.11±0.08, 0.01±0.01, 0.01±0.01 and 0.39±0.40, respectively) (p<0.0001). A high percentage of HLA-G +cells (30.47±26.75) was detectable on CD4dullCD8high cells from SSc patients only.ConclusionsThese data indicate that in SSc secretion and/or shedding of sHLA-G and mHLA-G are clearly elevated and involved in immune dysregulation.References[1] Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med2009;360:1989–2003.[2] Negrini S, Fenoglio D, Parodi A, Kalli F, Battaglia F, Nasi G, et al. Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis. Front Immunol2017;8:18.[3] Parel Y, Aurrand-Lions M, Scheja A, Dayer JM, Roosnek E, Chizzolini C. Presence of CD4+CD8+ double-positive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosis. Arthritis Rheum2007;56:3459–67.AcknowledgementsThis work was supported by “Gruppo Italiano Lotta alla Sclerodermia” (GILS). The sponsor had no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.Disclosure of InterestNone declared

Background Combination analgesics are effective in acute pain, and a theoretical framework predicts efficacy for combinations. The combination of dexketoprofen and tramadol is untested, but predicted to be highly effective. Methods This was a randomised, double-blind, double-dummy, parallel-group, placebo-controlled, single-dose trial in patients with moderate or severe pain following third molar extraction. There were ten treatment arms, including dexketoprofen trometamol (12.5 mg and 25 mg) and tramadol hydrochloride (37.5 mg and 75 mg), given as four different fixed combinations and single components, with ibuprofen 400 mg as active control as well as a placebo control. The study objective was to evaluate the superior analgesic efficacy and safety of each combination and each single agent versus placebo. The primary outcome was the proportion of patients with at least 50 % max TOTPAR over six hours. Results 606 patients were randomised and provided at least one post-dose assessment. All combinations were significantly better than placebo. The highest percentage of responders (72 %) was achieved in the dexketoprofen trometamol 25 mg plus tramadol hydrochloride 75 mg group (NNT 1.6, 95 % confidence interval 1.3 to 2.1). Addition of tramadol to dexketoprofen resulted in greater peak pain relief and greater pain relief over the longer term, particularly at times longer than six hours (median duration of 8.1 h). Adverse events were unremarkable. Conclusions Dexketoprofen trometamol 25 mg combined with tramadol hydrochloride 75 mg provided good analgesia with rapid onset and long duration in a model of moderate to severe pain. The results of the dose finding study are consistent with pre-trial calculations based on empirical formulae. Trial registration EudraCT (2010-022798-32); Clinicaltrials.gov ( NCT01307020 ).

BackgroundSkin fibroblasts (SFs) are involved in the excessive production of extracellular matrix (ECM) proteins which characterises fibrosis in systemic sclerosis (SSc).1 Myofibroblasts which are characterised by a higher expression of pro-fibrotic molecules (a-SMA: alpha-smooth muscle actin; S100A4: fibroblast-specific protein-1) as well as by the over-production of ECM proteins (FN: fibronectin; collagens type I and III), may originate from the activation and differentiation of resident fibroblasts after multiple profibrotic stimuli.2 CTLA4-Ig interacts with the cell surface costimulatory molecule CD86 and can downregulate the target cell activation.3 ObjectivesTo evaluate CD86 expression and the in vitro effects of CTLA4-Ig on skin fibroblasts (SFs).MethodsSkin biopsies were obtained from 8 “limited” cutaneous SSc patients (treated only with vasodilators, mainly cyclic prostanoids) and 4 healthy subjects (HSs), after EC and patient informed consent. After 8 days (T8) of culture, SFs obtained from biopsies were treated for 24 and 48 hours, in the absence or in the presence of CTLA4-Ig (10, 50, 100 and 500 micrograms/ml). Evaluation of CD86 expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, also human macrophages obtained from PBMCs of SSc patients, were cultured. The statistical analysis was carried out by the non-parametric Mann-Whitney U test.ResultsCultured SSc fibroblasts showed a very low gene expression level of CD86, compared to cultured macrophages of SSc patients, taken as positive control for CD86 expression (99% less). Therefore, cultured SSc fibroblasts treated for 24 hours and for 48 hours with CTLA4-Ig (10, 50, 100 and 500 micrograms/ml) did not show any significant modulation in the gene expression levels of CD86, compared to untreated fibroblasts (CNT). Interestingly, cultured HSs fibroblasts treated with CTLA4-Ig for 24 hours and for 48 hours showed a significant decrease in the gene expression of CD86, limited to the highest dose (500 micrograms/ml), compared to CNT (0.16% and 0.64% less, respectively) (p<0.05).ConclusionsIn the present short-term study (24 and 48 hours), no significant effects at qRT-PCR resulted after CTLA4-Ig treatment of cultured SSc SFs. The result might arise from a limited expression of CD86, as consequence of a retained advanced differentiation of the the SSc fibroblasts. On the contrary, a significant reduction of CD86 expression on HSs fibroblasts treated with CTLA-4Ig was observed.References[1] Asano Y. J Dermatol2017. Epub ahead of print. [Review].[2] Hin ZB, Phan SH, et al. Am J Pathol2007;170:1807–16.[3] Cutolo M, et al. Arthritis Res Ther2009;11:176–85.AcknowledgementsDisclosure of InterestM. Cutolo Grant/research support from: BMS, Actelion, Celgene, Boehringer, P. Montagna: None declared, S. Soldano: None declared, A. C. Trombetta: None declared, P. Contini: None declared, B. Ruaro: None declared, A. Sulli: None declared, S. Scabini: None declared, E. Stratta: None declared, R. Brizzolara: None declared

BackgroundIn the pathogenesis of systemic sclerosis (SSc), the immune cell activation is an important event that includes alteration in the macrophage polarisation.1 Macrophages may polarise into classically activated (M1), which are characterised by the expression of specific markers such as Toll-like receptors (TLR2 and 4) and costimulatory molecules (CD80 and CD86), or alternatively activated (M2), which are characterised by the expression of specific phenotype markers such as mannose receptor-1 (CD206) and macrophage scavenger receptors (CD204 and CD163). M2 are present in the circulation and in the skin infiltrate of SSc patients (pts), where they seem to contribute to fibrosis.2–4 ObjectivesTo characterise circulating M2 monocytes/macrophages from SSc pts and healthy subjects (HSs) by their co-expression of CD204, CD163 and CD206, as well as cells expressing both M1 and M2 phenotype markers.MethodsFifty-eight SSc pts (54 females/4 males, mean age 63±13 years), fulfilling the new EULAR/ACR criteria for SSc, and 27 age-matched HSs were consecutively enrolled after Informed Consent was obtained. Peripheral blood was collected and the antibodies CD14-APC-Vio770 and CD45-VioGreen were used to identify the monocyte/macrophage lineage; CD204-VioBright-FITC, CD163-PE-Vio770 and CD206-PeerCP-Vio700 were used to characterise the M2 phenotype, whereas CD80-APC, CD86-VioBlue, TLR4-PE and TLR2-PE-Vio615 were used to characterise the M1 phenotype (Miltenij Biothech). Flow Cytometry analysis was performed using Navios Flow Cytometer and the related Navios analysis software (Beckman Coulter).ResultsIn the CD14+cell subset (monocytes), the CD14+CD163+CD206+CD204+cell percentage was significantly increased in SSc pts compared to HSs (p=0.02). Inside the CD14+CD163+CD206+CD204+monocytes/macrophages a subset of cells co-expressing also TLR4, CD80 and CD86 was detected. This mixed population (M2/M1) of cells was significantly increased in SSc pts compared to HSs (p=0.003).At the same time, circulating monocytes/macrophages showing a full M2 phenotype and characterised as CD204+CD163+CD206+cells were investigated independently of the expression of CD14, and they also resulted significantly increased in SSc pts compared to HSs (p<0.0001).In the CD204+CD163+CD206+cell subset (M2), the percentage of cells expressing also TLR4, CD80 and CD86 (M1) was significantly increased in SSc pts compared to HSs (p<0.0001).ConclusionsThese results describe for the first time a subset of circulating cells belonging to the monocyte/macrophage lineage with a mixed phenotype, which are characterised by the expression of both M1 and M2 surface markers. These cells were observed to be increased in the peripheral blood of SSc pts compared to HSs, suggesting their possible role in the pathogenesis of the disease.References[1] Stifano G, et al. Curr Rheumatol Rep2016;18:2.[2] Higashi-Kuwata N, et al. Arthritis Res Ther2010;12:R128.[3] Wynn TA, et al. Immunol Rev. 2016;44:450–62.[4] Christmann RB, et al. Arhtitis Rheum. 2011;63:1718–28.Disclosure of InterestNone declared

BackgroundCirculating fibrocytes (CFs) are progenitor cells derived from bone marrow, expressing markers of both hematopoietic cells (CD45, MHC class II) and stromal cells (collagen I and III), together with the chemokine receptors, which regulate their migration into inflammatory lesions (CXCR4, CCR2, CCR7).1 CFs can migrate into SSc-affected tissues and can differentiate into fibroblasts/myofibroblasts.2 CFs express the CD86 (B7.2) costimulatory molecule and the adhesion molecules CD11a, CD54, ICAM-1, and CD58. The fusion molecule CTLA4-Ig interacts with CD86 and can downregulate the target cell.3 ObjectivesTo study the in vitro effects of CTLA4-Ig on cultured CFs.MethodsCFs were obtained from the peripheral blood samples of 8 “limited” cutaneous SSc patients (treated only with vasodilators, mainly cyclic prostanoids) and from 4 healthy subjects (HSs). CFs were characterised by fluorescence-activated cell sorter analysis (FACS), at basal time (T0) and after 8 culture days (T8), for CD45, collagen type I (COL I), CXCR4, CD14, CD86, and HLA-DRII expression. T8-cultured CFs were treated for 3 hours in the absence or in the presence of CTLA4-Ig (10, 50, 100 and 500 micrograms/ml). Quantitative real-time polymerase chain reaction (qRT-PCR) for CD86, COL I, IL1b, TGFb, αSMA, S100A4, CXCR2, CXCR4, CD11a were performed. The statistical analysis was carried out by the nonparametric Mann-Whitney U test. Skin samples for fibroblast (SFs) cultures were obtained from the same patients after EC and patient informed consent.ResultsAt qRT-PCR, T8-SSc fibrocytes, in the absence of CTLA4-Ig treatments, showed higher CD86 expression levels compared to HSs fibrocytes. Similarly also αSMA, S100A4, TGFb and COL I gene expression resulted higher in SSc fibrocytes compared to HSs. After CTLA4-Ig treatments, only in SSc fibrocytes, the αSMA and COL I gene expression resulted significantly decreased (p<0.01, p<0.05), whereas the gene expression for S100A4 resulted significantly increased (p<0.01), compared to untreated fibrocytes. Interestingly, in skin fibroblasts from the same SSc patients, the CD86 gene expression was found to be very low, compared to CFs.ConclusionsCirculating fibrocytes from patients affected by limited cutaneous SSc seem to be responsive and downregulated after in vitro CTLA4-Ig treatments, suggesting a possible antifibrotic effect on progenitor cells before their final homing and differentiation in active myofibroblasts. Fibroblasts from the same patient do not show the same expression of target molecules and reactivity to CTLA4-Ig.References[1] Bucala R. Mol Med2015;2:S3–5.[2] Keeleya EC, et al. The International Journal of Biochemistry & Cell Biology2010;42:535–42.[3] Cutolo M, et al. Arthritis Res Ther2009;11:176–85.Disclosure of InterestM. Cutolo Grant/research support from: BMS, Actelion, Celgene, Boehringer, P. Montagna: None declared, S. Soldano: None declared, A. C. Trombetta: None declared, B. Ruaro: None declared, P. Contini: None declared, A. Sulli: None declared, S. Scabini: None declared, E. Stratta: None declared, R. Brizzolara: None declared

Background Endothelial cell (EC) dysfunction and angiogenesis are involved in synovitis in established rheumatoid arthtritis (RA) [1]. Some studies reported that the ECs express the repertoire of costimulatory molecules including CD86 (B7.2), for adequate T cell interaction/activation [2]. Our recent data showed that the fusion protein CTLA4-Ig (abatacept), used as biological agent in RA therapy, already interacts with the CD86 molecule expressed in synovial cells [3,4]. Objectives In the present study CTLA4-Ig/CD86 interaction and VEGF-R and ICAM-1 protein expression was evaluated in vitro on activated ECs. Methods ECs (human microvascular endothelial cells, HMVEC, Lonza, Switzerland), after 7 days of culture, were induced by γ-IFN treatment (for 48 hours, 500 U/ml) to express CD86 molecules. Then, the cells were treated for 24 hours with CTLA4-Ig (10, 100, 500 μg/ml) and the protein expression of CD86, VEGF-R and ICAM-1 were evaluated by flow cytometric analysis (FACS). In addition, western blot analysis (WB) for VEGF-R and ICAM-1 protein expression were performed. Results FACS analysis showed that, after CTLA4-Ig treatment (10, 100, 500 μg/ml), activated ECs decreased their CD86-positivity (66%, 59% and 51%, respectively), compared to untreated cells (68%), suggesting a CTLA4-Ig/CD86 interaction and masking on their surface. Therefore, almost all the activated ECs expressed VEGF-R (79%) and all activated ECs strongly expressed ICAM-1 (99%). After 24 hours of CTLA4-Ig treatment, the cells showed a decrease dose-dependent in the mean fluorescence value for ICAM-1, while VEGF-R positivity resulted unchanged. WB analysis for VEGF-R and ICAM-1 protein expression also showed a marked decrease in activated ECs treated with CTLA4-Ig at 500 μg/ml. Conclusions The results observed at FACS analysis suggest an interaction between CTLA4-Ig and CD86 on activated ECs (expressing the CD86 molecule). In addition, a modulation in the expression of molecules relevant for the inflammatory and angiogenetic processes was observed. ICAM-1 protein expression shows on activated ECs a dose-dependent decrease after CTLA4-Ig treatment, at least for the higher dose treatment. Otherwise, VEGF-R protein expression showed a decrease only observed in WB analysis. In conclusion, ECs might be considered a further target for CTLA4-Ig in inflammatory conditions such as in presence of synovitis. References Marrelli A et al. Autoimmun Rev. 2011;10(10):595-8. Kreisel D et al. J Immunol 2002;169(11):6154-61. Brizzolara R et al. Reumatismo. 2011;63(2):80-54. Cutolo M et al. Clin Exp Rheumatol. 2013;31(6):943-6. Disclosure of Interest M. Cutolo Grant/research support: Bristol Myers Squibb, P. Montagna: None declared, S. Soldano: None declared, B. Seriolo Grant/research support: Bristol Myers Squibb, P. Contini: None declared, B. Villaggio: None declared, R. Brizzolara: None declared DOI 10.1136/annrheumdis-2014-eular.3563

Background Vascular endothelium is involved in several immune mediated diseases and in chronic inflammation. An important matter of debate is whether endothelial cells (ECs), both in resting or activated state, express the repertoire of costimulatory molecules such as CD86 (B7.2), for adequate T cell interaction/activation [1,2]. Objectives In order to evaluate the CD86 presence on ECs of different origin, we studied in vitro two distinct human ECs lines, both resting or activated with different immune/inflammatory stimuli (γIFN, IL-17, IL-1β). Methods Human Umbilical Vein Endothelial Cells (HUVEC, Lonza, Switzerland) and Human Microvascular Endothelial Cardiac Cells (HMVEC-C, Lonza, Switzerland), at fourth culture passage, were stimulated for 48 hours with γIFN (500 U/ml, Sigma, Milan, Italy) in order to activate them [3]. In addition, HMVEC-C were stimulated for 48 hours with two further stimuli: human IL-1β (500 U/ml, Adipogen, Incheon, South Korea) or IL-17 (100 ng/ml, Biovision, CA, USA). Thus, in both HUVEC and HMVEC-C, the expression of EC phenotypic markers was detected by flow cytometric analysis, using PE anti-human CD31 and CD105 mouse antibodies (Milteny Biotec Inc, CA, USA). In addition, the expression of CD86 in resting or activated ECs was detected by flow cytometric analysis, using a FITC anti-human CD86 mouse antibody (BD, Biosciences, NY, USA). In every three experiments specific isotype control was included. Results At flow cytometric analysis, the phenotypic characterization of the ECs by CD31 and CD105 specific staining was confirmed. CD31 and CD105 markers were found in respectively 99% and 96% of the unstimulated HUVEC, as well as in 93% and 96% of the unstimulated HMVEC-C. ECs in resting condition, expressed mild level of CD86 (57% on HUVEC and 60% on HMVEC-C) at 48 hours. The same ECs after 48 hours of γIFN stimulation, showed an evident increase of the fluorescence for CD86 expression (85% on HUVEC and 68% on HMVEC-C). Further evaluations limited to HMVEC-C, showed that IL-1β stimulation did not change the percentage of CD86 positive cells (61%) after 48 hours, compared with untreated cells (60%). Conversely, IL-17 treatment induced a light increase of CD86 positive cells (71%) after 48 hours, compared to untreated cells (60%). Conclusions Our study shows that ECs (both HUVEC and HMVEC) express constitutively the costimulatory molecule CD86, and the expression is increased in activated state. In particular, microvascular ECs activated with γIFN and IL-17, but not with IL-1β, show an increased CD86 expression compared to their resting condition. These results suggest the involvement and contribution of ECs during the immune/inflammatory response for example by facilitating T cell adhesion and migration. References Kreisel D et al. J Immunol 2002;169(11):6154-61. Lozanoska-Ochser et al. J Immunol 2008;181;6109-6116. Batten P et al. Immunol 1996;87:127-133. Disclosure of Interest None Declared

Background In rheumatoid arthritis (RA) the combination of glucocorticoids (GC) and the fusion protein CTLA4-Ig (abatacept) allows to obtain better clinical improvement than CTLA4-Ig monotherapy. Our recent data showed that CTLA4-Ig interact with the CD86 molecule and directly dowregulates RA synovial macrophages, inducing a reverse signaling upon the binding [1-3]. Objectives In this study the anti-inflammarory effect of dexamethasone (DEX) alone or combined with CTLA4-Ig was evaluated in vitro on cultured human activated macrophages. Methods THP-1 cells, activated into macrophages (PMA 0.05 μg/ml; 24 hrs), were cultured for 48 hrs with DEX (10–7 M) alone, or combined with CTLA4-Ig (500 μg/ml). Cells untreated and treated with CTLA4-Ig alone, were used as controls. CD86 expression was evaluated by immunofluorescence (IF) and by flowcytometry (FACS) analysis. IL-1β, TNFα and IL-6 protein production was investigated at 48 hrs after treatments by immunocytochemistry (ICC) and western blot analysis (WB). In addition, cytokine gene expression was evaluated at 1 and 3 hrs after treatments by quantitative real time polymerase chain reaction (qRTPCR). Results Qualitative IF of CD86 demonstrated a decreased expression on untreated macrophages after DEX treatment alone and, more prominently after DEX and CTLA4-Ig-combined treatment. Quantitative FACS analysis confirmed these results: DEX alone induced a reduction of CD86 expression on cells by 78% and DEX combined with CTLA4-Ig induced an evident CD86 decrease by 97%, compared to the untreated cells. ICC analysis showed a decrease in IL-1β and TNFα production in macrophages treated for 48 hrs with DEX alone (ns) or combined with CTLA4-Ig (p<0.05), compared to untreated cells. CTLA4-Ig alone also significantly reduced IL-1β and TNFα production (p<0.05). Otherwise, IL-6 production in macrophages treated for 48 hrs with DEX alone or combined with CTLA4-Ig resulted unchanged, compared to untreated cells. WB analysis confirmed these results obtained after 48 hrs of treatment. qRTPCR showed in macrophages treated with DEX alone or in CTLA4-Ig-combined treatment, after only 1 hr, a reduction for the expression of all assayed cytokine genes. CTLA4-Ig alone reduced IL-1β, TNFα and IL-6 expression at 3 hrs, but not at 1 hr from treatment. At 3 hrs from DEX and DEX plus CTLA4-Ig treatment, cells still showed a reduction limited to IL-1β and IL-6 gene expression. Conclusions Both DEX and DEX plus CTLA4-Ig treatments, induce a reduction in CD86 expression and an anti-inflammatory effect on human activated macrophages, by decreasing both cytokine protein and gene expression. The results seem mainly due to the CTLA4-Ig/CD86 binding and also partially related to the genomic effects exerted by DEX on cytokine gene expression. Results might explain the improved clinical conditions observed in RA patients treated with CTLA4-Ig and GC (1,4). References Brizzolara R et al. Reumatismo. 2011;63(2):80-54. Cutolo M et al. Clin Exp Rheumatol. 2013;31(6):943-6. Bonelli M et al. Arthritis Rheum 2013;65(3):599-607. Cutolo M et al. Ann N Y Acad Sci 2010;1193:15-21 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2844

BackgroundIn systemic sclerosis (SSc) tissues, the immune inflammatory infiltrate primarily consists of macrophages and T cells (1). Within the macrophage population, alternative activated macrophages (M2) are characterized by the expression of specific phenotype markers, including mannose receptor1 (CD206) and scavenger receptors (CD204, CD163 and CD36). M2 have been shown to participate in the fibrotic process as major producers of TGFβ (1). Increased levels of endothelin-1 (ET-1) have been shown in serum of SSc patients (2). Moreover, ET-1 may be considered one of the possible links between vascular damage and fibrotic process in SSc (3,4).ObjectivesTo investigate the presence of M2 in the peripheral blood (PB) and skin of SSc patients, as well as the ability of ET-1 to induce in vitro the transition into M2 of PB mononuclear cells (PBMCs) from healthy subjects (HS).MethodsEight patients with limited cutaneous involvement (lSSc, mean age 65±7 yrs), who fulfilled the new EULAR/ACR criteria (5), and five age-matched HS were enrolled into the study after obtaining their informed consent and Ethical Committee approval. In all lSSc patients and HS whole blood was collected for PBMC isolation and skin biopsy was performed for the evaluation of immune-inflammatory infiltrate. CD206 and CD204 expressions were analysed in PBMCs and skin biopsies by flow cytometry (FC) and immunohystochemistry (IHC), respectively. The expression of CD14 (marker of monocytes lineage) and CD68 (marker of activated macrophages) was also evaluated by FC and IHC. PBMC-derived monocytes isolated from HS (2x106 cells/ml), after 12 hrs in growth medium (with 10% of fetal bovine serum) were treated for 72hrs with ET-1 (100nM). Untreated cells were used as controls. The expression of CD206, CD204 and CD68 was evaluated by ICC. Statistical analysis was performed by Mann-Whitney non-parametric U-test.ResultsSSc patients showed a significantly higher percentage of CD206+/CD14+cells in the PBMC population compared to HS (p<0.01). These cells also co-expressed CD204 as observed by FC analysis. CD206+ and CD204+cells were also detected in the immune inflammatory infiltrate of the skin of SSc patients. Conversely, in the HS skin no CD206+and CD204+cells were observed. Of note, ET-1 induced the transition of PBMCs from HS into activated macrophages and the expression of CD206 and CD204.ConclusionsPreliminary results show an increased circulating percentage of the CD206+/CD14+cell subset (characterised by the co-expression of CD204) in SSc patients, suggesting that they might belong to the M2 lineage. The presence of CD206+ and CD204+cells (M2) in the immune inflammatory infiltrate of skin might also suggest a possible role of these cells in SSc. Finally, ET-1 seems to be implicated as enhancer of the monocyte activation and polarization into profibrotic M2.ReferencesMantovani A et al.J Pathol.2013;229:176-85.Sulli A et al.J Rheumatol.2009;36:1235-39.Leask A.Pharmacol Res.2011;63:502-3.Cutolo M et al.J Rheumatol.2013;40:40-5.van den Hoogen F et al.ArthritRheum.2013;65:2737-47.Disclosure of InterestNone declared